Prevention of nsaid enteropathy with microbiota-derived tryptophan-metabolite

ABSTRACT

This disclosure relates to methods and compositions for addressing conditions of dysbiosis and/or inflammation, such as enteropathy, associated with administration of non-steroidal anti-inflammatory drug (NSAID). The disclosure includes methods comprising administering an effective amount of a tryptophan derived microbiota metabolite (TDMM) to a subject that has also been administered, or is expected to have administered, a NSAID.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of Provisional Application No.62/310,606, filed Mar. 18, 2016, Provisional Application No. 62/310,648,filed Mar. 18, 2016, Provisional Application No. 62/310,643, filed Mar.18, 2016, and Provisional Application No. 62/310,630, filed Mar. 18,2016, each of which is expressly incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under GM106251 awardedby The National Institutes of Health, National Institute of GeneralMedical Sciences; AI110642 awarded by The National Institutes of Health,National Institute of Allergy and Infectious Disease; A095788 awarded byThe National Institutes of Health, National Institute of Allergy andInfectious Disease; and MCB-1120827, awarded by the National ScienceFoundation. The Government has certain rights in the invention.

FIELD OF THE INVENTION

This disclosure relates to methods and compositions for addressingconditions of dysbiosis and/or inflammation, such as enteropathy,associated with administration of non-steroidal anti-inflammatory drug(NSAID).

BACKGROUND

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the mostfrequently used medications worldwide for routine relief of pain orfever, to manage various forms of arthritis and inflammatory intestinaldisorders, and to prevent or treat alimentary cancers. Despite theireffectiveness for managing these varied and highly prevalent conditions,NSAIDs cause damage to the gastrointestinal (GI) tract. Although methodsfor diagnosis and effective treatment of NSAID-induced lesions of theproximal GI tract (i.e., gastropathy) have been well documented, thepathogenesis, diagnosis, and treatment of NSAID-induced damage of the GItract distal to the duodenum (known as NSAID enteropathy, primarilyaffecting the distal jejunum and ileum) remain unclear. The magnitude ofthe problem of NSAID enteropathy is alarmingly high. In the UnitedStates, NSAID enteropathy results in approximately 100,000hospitalizations and 16,500 deaths each year. Additionally, two-thirdsof both short- and long-term NSAID users develop distal small intestinallesions. Although the use of either NSAIDs considered to be safer forthe GI tract or other ancillary therapies have reduced the incidence andseverity of NSAID-induced gastropathy, the incidence of NSAIDenteropathy has remained constant or has increased.

In view of the foregoing, there is a need for compositions and methodsto ameliorate or prevent side effects, such as enteropathy, associatedwith NSAID administration. The present disclosure addresses this andrelated needs.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the disclosure provides a method of treating dysbiosis ofcommensal microbiota in a subject. The method comprises administering aneffective amount of a tryptophan derived microbiota metabolite (TDMM),or a precursor, prodrug, or acceptable salt thereof, to a subject inneed thereof.

In one embodiment, the subject has received or is expected to receiveadministration of an agent suspected to cause dysbiosis. In oneembodiment, the agent causes a reduction in a gram-positive component ofthe microbiota. In one embodiment, the agent causes an increase in agram-negative component of the microbiota. In one embodiment, the agentis a non-steroid anti-inflammatory drug (NSAID). In one embodiment, theTDMM is indole. In one embodiment, the TDMM, or a precursor, prodrug, oracceptable salt thereof, is co-administered with the agent. In oneembodiment, the TDMM, or a precursor, prodrug, or acceptable saltthereof, is administered in a pharmaceutical composition that alsocomprises the agent. In one embodiment, the TDMM is administered byintra-peritoneal (IP), intravenous (IV), topical, parenteral,intradermal, transdermal, oral (e.g., via liquid or pill), orrespiratory (e.g., intranasal mist) routes. In one embodiment, thesubject is a mammal, such as a human or rodent. In one embodiment,treating the dysbiosis prevents or ameliorates enteropathy.

In another aspect, the disclosure provides a method of treatingenteropathy associated with NSAID in a subject. The method comprisesadministering an effective amount of a tryptophan derived microbiotametabolite (TDMM), or a precursor, prodrug, or acceptable salt thereof,to a subject in need thereof. In one embodiment, the NSAID causes areduction in a gram-positive component of the microbiota. In oneembodiment, the NSAID causes an increase in a gram-negative component ofthe microbiota. In one embodiment, the NSAID is selected from aspirin,salsalate, celecoxib, diclofenac, etodolac, ibuprofen, indomethacin,ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam,sulindac, meloxicam, tolmetin, and the like. In one embodiment, the TDMMis indole. In one embodiment, the effective amount of indole is at leastabout 5 mg/kg. In one embodiment, the TDMM, or a precursor, prodrug, oracceptable salt thereof, is co-administered with the NSAID. In oneembodiment, the TDMM, or a precursor, prodrug, or acceptable saltthereof, is administered in a pharmaceutical composition that alsocomprises the NSAID. In one embodiment, the TDMM is administered byintra-peritoneal (IP), intravenous (IV), topical, parenteral,intradermal, transdermal, oral (e.g., via liquid or pill), orrespiratory (e.g., intranasal mist) routes. In one embodiment, thesubject is a mammal, such as a human or rodent.

In another aspect, the disclosure provides a method of treating acondition characterized by inflammation in the GI tract. The methodcomprises administering an effective amount of a tryptophan derivedmicrobiota metabolite (TDMM), or a precursor, prodrug, or acceptablesalt thereof, to a subject in need thereof. In one embodiment, theinflammation is associated with administration of an NSAID to thesubject.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising: at least one TDMM, or a precursor, prodrug, or acceptablesalt thereof; at least one NSAID composition, or a precursor, prodrug,or acceptable salt thereof and a pharmaceutically acceptable carrier. Inone embodiment, the TDMM is indole.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIGS. 1A-1F illustrate that indole attenuates severity of non-steroidalanti-inflammatory drug (NSAID) enteropathy. FIG. 1A graphicallyillustrates fecal calprotectin levels were determined by enzyme-linkedimmunosorbent assay (ELISA) on fecal samples on Day 0 (white bar) andthen again after 6 days of therapy (black bar) with indomethacin,indole, or the combination. Day 6 fecal calprotectin values weresignificantly different from day 0 calprotectin only for the animalsthat received indomethacin alone (P=0.0095). FIG. 1B graphicallyillustrates microscopic pathology scores from analysis of Swiss-rolled H& E stained small intestinal sections (n=5/group). Error bars represent95% confidence intervals about the mean score. Groups with differentletters differed significantly (P<0.05): control and indole-treated miceare not different from one another; NSAID alone results in a significantdifference from control, indole alone, and from NSAID+indole; theNSAID+indole group was significantly higher than control and indolealone groups, but significantly lower than the NSAID group. FIG. 1Cshows representative H & E stained sections of small intestine. Themicroscopic appearance of both the control and indole appear normal,whereas the small intestine of NSAID-treated mice showed ulceration,inflammation in the lamina propria and epithelium, and thickening andblunting of the villi. These pathological findings were ameliorated byco-administration of indole with NSAID (i.e., NSAID+indole). FIG. 1Dgraphically illustrates the ratio of villus height to crypt depth takenfrom small intestinal mucosa; relative to the controls, this ratio wasonly significantly decreased for mice in the NSAID group. FIG. 1Egraphically illustrates the percentage of CD11b- and GR-1-positive cells(i.e., neutrophils) in the spleen after 7 days of treatment. Groups withdifferent letters differed significantly. Indole significantlyattenuated the NSAID-induced increase in splenic neutrophils. FIG. 1Fgraphically illustrates the percentage of CD11b positive andGR-1-positive cells (i.e., neutrophils) in the mesenteric lymph nodes(MLN) after 7 days of treatment. Groups with different letters differedsignificantly. Indole significantly attenuated the NSAID-inducedincrease in MLN neutrophils.

FIGS. 2A-2F illustrate that indole increases abundance of Clostridialesand prevents NSAID-induced shift of the microbiota and inferredmetagenome. FIG. 2A illustrates a principal component analysis (PCA)plots of 16S rRNA sequencing of the fecal microbiota at the phylum levelwith biplot overlay and analysis of similarity (ANOSIM) based on theunweighted Unifrac distance metric (located in the lower right quadrant)of the operational taxonomic unit (OTU) table from day 0 revealed nosignificant differences among the groups at day 0.The area of the grayshaded shapes reflects the variation among individuals in a group: thelarger the shaded area, the more variation among the individuals in thatgroup. FIG. 2B illustrates a principal component analysis (PCA) plots of16S rRNA sequencing of the fecal microbiota at the phylum level withbiplot overlay and ANOSIM based on the unweighted Unifrac distancemetric (located in the lower right quadrant) of the OTU table from day 7reveal that there is a significant difference among the groups at day 7.After 7 days, the NSAID group is significantly shifted from the other 3groups, and this shift was associated with a qualitative increase in thephylum Bacteroidetes and loss of Firmicutes. FIG. 2C graphicallyillustrates the abundance of Clostridales from Day 0 (orange) and Day 7(green) and Bacteroidales Day 0 (white) and Day 7 (black). Error barsrepresent the upper bound of the symmetrical 95% confidence intervalsabout the mean of the 5 animals in each group and P values displayed onthe graph are corrected for multiplicity of comparisons by the method ofSidak. Clostridiales were significantly increased on Day 7 relative toDay 0 by co-administration of indole with the NSAID indomethacin. FIG.2D graphically illustrates the mean abundance of several families ofClostridiales at Day 0 and Day 7 for each of the treatment groupsdepicting reduction of these families following NSAID therapy butexpansion indole is co-administered. FIG. 2E illustrates PCA plots ofinferred metagenome of the fecal microbiota from Day 0. ANOSIM based onthe Bray Curtis distance measure revealed no significant differenceamong the groups at day 0. FIG. 2F illustrates PCA plots of inferredmetagenome of the fecal microbiota from Day 7. ANOSIM based on the BrayCurtis distance measure revealed that, consistent with the microbiotadata, the NSAID group visually separated from the other groups; however,this apparent difference was not significant (P>0.05 by ANOSIM based onBray Curtis dissimilarity measure).

FIGS. 3A-3D illustrate that NSAID enteropathy results in upregulation ofinnate immune response pathways and the co-administration of indoleregulates this response. FIG. 3A is a heatmap showing upregulated (red)and down regulated (blue) pathways in the small intestinal mucosa fromNSAID-treated mice compared with control animals. FIG. 3B is a 2-columnheatmap showing upregulated (red) and down-regulated (blue) pathways inthe small intestinal mucosa from NSAID-treated mice compared withcontrol mice (left column) and NSAID+indole versus control mice (rightcolumn). FIG. 3C is a heatmap of the Z-scores of the canonical pathwaysto which the differentially expressed genes between control vs.NSAID-treated animals (left column) and control vs. NSAID+indole (rightcolumn) were mapped. FIG. 3D is a heat map of the Z-scores of theupstream regulators to which the differentially expressed genes betweencontrol vs. NSAID-treated animals (left column) and control vs.NSAID+indole (right column) were mapped.

FIG. 4 schematically illustrates that co-administration of indole withindomethacin attenuates NSAID enteropathy and indole may exert thesebeneficial effects through several possible mechanisms. NSAIDenteropathy is characterized by a loss of barrier function resulting inan influx of luminal contents and a massive innate immune response.Indole is known to have direct effects on intestinal epithelial cellsincluding upregulation of tight-cell junctional proteins, effects onimmune responses including inhibition of nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κB), and regulatingthe overall innate immune response. Moreover, co-administration ofindole with NSAID administration resulted in an increase in theabundance of Firmicutes, principally Costridales, known to be importantin maintaining intestinal homeostasis, and appeared to prevent anyincrease in Bacteroidales.

FIGS. 5A and 5B illustrate the noninvasive identification of 6 key genesaltered between NSAID and NSAID+Indole treatments. FIG. 5A is a networkof the top 15 upstream regulator, identified by 2-feature LDA in thecontext of NSAID vs control animals with FDR P-values and fold changesoverlaid. FIG. 5B is the same network with P-values and fold changes ofNSAID+indole overlaid reveal 11 genes that were altered (oppositefold-change either realized or predicted) by the co-administration ofindole.

FIGS. 6A and 6B illustrate that Indole attenuates small intestinal muralthickening associated with NSAID enteropathy. FIG. 6A is a diagramshowing the location of representative measures for calculating villusheight, crypt depth, and mural thickness (indicated in black lines).Three sets of measurements from three separate sections were recordedfor each animal by an observer blinded to treatment group. FIG. 6Bgraphically illustrates representative mural thickness among all groups.

FIGS. 7A and 7B illustrate that co-administration of indole reducesNSAID-induced neutrophilic infiltration. FIG. 7A provides representativedot-plots of double-positive CD11b/GR-1 cells (neutrophils) from spleen;and FIG. 7B provides mesenteric lymph nodes.

FIGS. 8A-8D. FIG. 8A graphically illustrates alpha rarefaction curvesfor each treatment group showing numbers of observed species (richness)at each sampling depth on the y-axis and sequences/sample on the x-axisup to a sampling depth of 10,800 sequences/sample at Day 0. FIG. 8Bgraphically illustrates the alpha rarefaction curves at Day 7. FIG. 8Cgraphically illustrates estimated Good's coverage at an even samplingdepth of 10,800 reads/sample at Day 0. FIG. 8D graphically illustratesestimated Good's coverage at Day 7.

FIGS. 9A and 9B. FIG. 9A graphically illustrates abundance of members ofthe phylum Bacteroidetes in fecal 16S rRNA sequences on Day 0 (whitebar) and Day 7 (black bar). FIG. 9A graphically illustrates abundance ofmembers of the phylum Firmicutes in fecal 16S rRNA sequences on Day 0(white bar) and Day 7 (black bar).

FIGS. 10A and 10B are bar graphs of log fold-change and false discoveryrate (FDR) P value of several pro-inflammatory cytokines (FIG. 10A) andchemokines (FIG. 10B) from NSAID+indole versus control mice (white bar)and NSAID vs. control animals (black bar) from the small intestinalmucosal transcriptome after 7 days of therapy.

DETAILED DESCRIPTION

The pathophysiology of NSAID enteropathy is complex and incompletelyunderstood. It appears to involve deleterious effects of NSAIDs on theintestinal mucosa including enterocyte cell death, increased mucosalpermeability, and interaction of the damaged mucosa with luminalcontents including bacteria (GI microbiota) and bacterial products orcomponents such as lipopolysaccharide (LPS). The GI microbiota has beenimplicated as an important contributor to NSAID enteropathy. Forexample, administration of NSAIDs causes a dysbiosis characterized by areduction of the predominately gram-positive phylum Firmicutes and acorresponding increase of gram-negative bacteria. Germ-free rats lackingintestinal microbiota do not develop NSAID enteropathy, whereas theydevelop NSAID-induced intestinal lesions when colonized withgram-negative bacteria. Concurrent administration of NSAIDs andantimicrobials targeting gram-negative bacteria reduces the severity ofNSAID-induced gastrointestinal lesions in rats.

In view of the role of some components of the microbiota in exacerbatingNSAID enteropathy, the inventors investigated the whether there might bemicrobiota-derived metabolites that could mitigate gut inflammation andother pathology that characterizes NSAID enteropathy. As described inmore detail below, the inventors examined the interaction of the host,the NSAID indomethacin, and a microbiota-derived metabolite, indole, ina murine model of NSAID enteropathy. The inventors surprisinglydemonstrated that co-administration of indole attenuated NSAID-inducedintestinal damage via a number of classic metrics. Notably, theinventors observed that co-administration of indole prevented theclassic NSAID-induced shift of the microbiota, an important aspect ofthe pathophysiology of NSAID enteropathy. Additionally, the inventorsperformed RNA-sequencing of the small intestinal mucosa (representing anin vivo analysis of the transcriptome of NSAID enteropathy) and examinedhow the co-administration of indole altered this transcriptome. Thisdataset is available to the public (NCBI accession number PRJNA290483).

Accordingly, in view of the foregoing, in one aspect the disclosureprovides a method of treating dysbiosis of commensal microbiota in asubject. The method comprises administering an effective amount of atryptophan derived microbiota metabolite (TDMM), or a precursor orprodrug thereof.

As used herein, the term “treating” means preventing, reducing, slowing,or ameliorating symptoms associated with the indicated condition.

As used herein, the term “dysbiosis” refers to an imbalance of thediverse microbial community that inhabits the intestinal tract of asubject (e.g., vertebrate host). The commensal microbial community isalso referred to herein as “microbiota”. In some embodiments, thedysbiosis leads to or is correlated with enteropathy. As used herein,the term “enteropathy” refers to the presence of lesions in the regionof the gastrointestinal (GI) tract that is generally distal to theduodenum. In some embodiments, this region includes at least a portionof the distal jejunum and ileum. Accordingly, in some embodiments ofthis aspect, treating dysbiosis refers to preventing, reducing, slowing,or ameliorating the symptoms associated with enteropathy. In someembodiments, the enteropathy is associated with the agent, such as withadministration of an NSAID.

In some embodiments, the dysbiosis is associated with inflammationand/or other pathologies similar to enteropathy, such as experienced indisorders such as colitis, inflammatory bowel disease (IBD), psoriasis,rheumatoid arthritis, multiple sclerosis, and the like. While themethods generally disclosed herein are described mostly in the contextof NSAID enteropathy, it is known that several signaling target thathave a role in enteropathy, such as stat3, akt, and mTor, also play arole in such other inflammatory diseases. Accordingly, such otherdiseases and conditions are encompassed by the present disclosure.

As used herein, the term “effective amount” or “therapeuticallyeffective” amount refers to a sufficient quantify or concentration ofthe agent, e.g., TDMM, so as to elicit an intended result, such asprevention or reduction of dysbiosis. Effective amounts can be readilydetermined and optimized per normal

As used herein, the term “tryptophan derived microbiota metabolite”(“TDMM”) refers to metabolites produced by microorganisms of thecommensal microbiota that resides in the intestinal tract. Theindividuals of the microbiota inhabit the space of the intestines andexist in homeostasis with the healthy vertebrate host. Thus, presumably,the metabolite products produced by the individuals of the microbiotahave been selected over time to promote a stable environment beneficialto both the microbiota and host. For example, such beneficialenvironment is one that is adverse to colonization by pathogens or thatavoid inflammatory conditions in the mucosa. Specific metabolitesreferred to by the term TDMM are derivatives of tryptophan and includecompounds such as indole, hydroxyindole (e.g., 2-hydroxyindole, 3-hydroxyindole, 5-hydroxyindole, 7-hydroxyindole),1-(2-carboxyphenylamino)-1-deoxy-D-ribulose-5-phosphate,5-Hydroxy-L-tryptophan, indoleglycerol phosphate, indolepyruvate,N-(5-Phospho-D-ribosyl)anthranilate, tryptamine, indole-3-acetate,L-formylkynurenine, L-Tryptophanyl-tRNA(Trp), indole-3-acetamide,indole-3-pyruvate, indole-3-lactic acid, tryptophol,indole-3-acetaldehyde, indole-3-aldehyde, isatin (indole-2,3-dione),isoindigo, indirubin, indoxyl-sulfate, and 2-oxyindole. The TDMMencompasses such molecules that have been directly obtained frommicrobiota individuals. Such TDMMs can be isolated, purified, orpartially purified through well-established methods. However, the termTDMM, while referring to the feature of a “microbiota metabolite,” isnot necessarily limited to the specific source of one or more individualorganisms from the commensal microbiota. Instead, it merely refers tothe fact that commensal microorganisms of the microbiota are known toproduce such a compound. Thus, the TDMM used as part of this disclosurecan also be obtained from an organism that is not typically consideredto be a member of a commensal microbiota of a vertebrate organism(whether the vertebrate individual is the source of the naïve T cell ornot). Instead, the TDMM can be produced by known methods, such astypical recombinant approaches, in preferred laboratory strains ofbacteria, and the like. Furthermore, the TDMM can be producedsynthetically, as appropriate through known methods of syntheticchemistry. In any of the above embodiments, the subject can beadministered with a composition that is a precursor to or prodrug of anyof the above listed TDMM compound embodiments.

In one embodiment, the TDMM is indole, which is typically representedwith the following structure:

In some embodiments, the subject has received or is expected to receivean administration of an agent, e.g., drug, with a suspected side-effectof causing dysbiosis. As described above, some agents associated withdysbiosis have been shown to result in the reduction of gram-positivebacteria in the phylum Firmicutes. Accordingly, in some embodiments, theagent is known to cause a relative decrease in a gram-positive componentof the microbiota, such as bacteria in the phylum Firmicutes. In someembodiments, the agent is known to cause a relative increase in agram-negative component in the microbiota.

In some embodiments, the agent is a nonsteroidal anti-inflammatory drug(NSAID).

The term “nonsteroidal anti-inflammatory drug” or the abbreviation“NSAID” refers to a class of drugs that are typically used for analgesicand antipyretic purposes, although they are also used in higher dosesfor anti-inflammatory effects. Due to non-addictive characteristics,NSAIDs are often used as alternatives to narcotic drugs. Most NSAIDsinhibit the activity of cyclooxygenase-1 (COX-1) and cyclooxygenase-2(COX-2), and thereby, the synthesis of prostaglandins and thromboxanes.It is thought that inhibiting COX-2 leads to the anti-inflammatory,analgesic and antipyretic effects. Most relevant to the presentdisclosure are NSAIDs that cause adverse reactions in the intestinaltract, especially dysbiosis in the microbiota and enteropathy.Exemplary, non-limiting NSAIDs relevant to the present disclosure areset forth in TABLE 1.

TABLE 1 Exemplary, non-limiting list of NSAIDs relevant to the presentdisclosure NSAID category Examples Salicylates Aspirin (acetylsalicylicacid) Diflunisal (Dolobid) Salicylic acid and other salicylatesSalsalate (Disalcid) Propionic acid Ibuprofen^([64]) derivativesDexibuprofen Naproxen Fenoprofen Ketoprofen Dexketoprofen FlurbiprofenOxaprozin Loxoprofen Acetic acid Indomethacin derivatives TolmetinSulindac Etodolac Ketorolac Diclofenac Aceclofenac Nabumetone Enolicacid Piroxicam (Oxicam) Meloxicam derivatives Tenoxicam DroxicamLornoxicam Isoxicam Phenylbutazone (Bute) Anthranilic acid Mefenamicacid derivatives Meclofenamic acid (Fenamates) Flufenamic acidTolfenamic acid Selective COX-2 Celecoxib inhibitors Rofecoxib (Coxibs)Valdecoxib Parecoxib (FDA withdrawn, licensed in the EU) Lumiracoxib(TGA cancelled registration) Etoricoxib (not FDA approved, licensed inthe EU) Firocoxib (used in dogs and horses) Sulfonanilides NimesulideOthers Clonixin Licofelone (5-LOX/COX inhibitor) H-harpagide (in Figwortor Devil's Claw)

Temporally, the TDMM can be administered prior to, concurrently with, orafter administration of the agent. It is within the skill of theordinary artisan to determine and optimize the most efficacious regimenof pre-, co-, and/or post-administration of the TDMM relative to theagent. Furthermore, it will be appreciated that multiple administrationsof the TDMM relative to one or more exposures to the agent areencompassed by the present disclosure. Again, determining and optimizingsuch administration regimens is within the skill of the ordinaryartisan.

Administration of the TDMM can be in any appropriate route ofadministration. For example, the TDMM can be administered byintra-peritoneal (IP), intravenous (IV), topical, parenteral,intradermal, transdermal, oral (e.g., via liquid or pill), rectal, orrespiratory (e.g., intranasal mist) routes. In preferred embodiments,the TDMM is ingested, e.g., via liquid or pill, etc. to facilitatedelivery of the TDMM to the intestinal tract where the microbiotareside.

The effective amount of TDMM, or a precursor or prodrug thereof, candepend on factors pertaining to the subject and the route ofadministration, etc., and can be readily determined according to theskill of the ordinary artisan. In some embodiments, a singleadministration (of potentially one or multiple administrations)comprises at least about 1 mg/kg, 5 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg,30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100mg/kg, or more. Dosing can be readily determined for any particularsubject based on routine cell and animal toxicity studies so as to avoiddeleterious effects of high concentrations, while still achieving thedesired effect.

In some embodiments, the microbiota of the subject is induced to produceelevated levels of the appropriate TDMM, such as indole. In this regard,additional microbiota organisms that are known to produce high levels ofthe TDMM can be administered to the subject. In some embodiments, theadministered microbiota organisms can be artificially selected orgenetically engineered to produce higher levels of the TDMM whenresiding in the GI tract of the subject. For example, in someembodiments the genetically engineered microorganisms are engineered toexpress, stably or transiently, higher levels of a tnaA gene, whichencodes the enzyme responsible for tryptophan metabolism and productionof indole. Alternatively or additionally, the subject can be providedwith elevated levels of tryptophan, such as in dietary supplements, soas to allow the microbiota to produce higher levels of TDMMs.

The subject can be any organism for which maintenance of a balance ofcommensal microbiota in the intestinal tract is desired. The subject canbe any vertebrate, including birds (e.g., chickens) and mammals,including primates (such as humans); rodents (such as mouse, rat, andguinea pig); cat; dog; cow; horse; sheep; pig; and the like.

In another aspect, the disclosure provides a method of treating aninflammatory disease or condition. In some embodiments, the condition ischaracterized by inflammation in the gut. In some embodiments, thedisease or condition is enteropathy associated with NSAID (i.e.,“NSAID-enteropathy”) in a subject. As described above, other diseases orconditions involve inflammation and/or other pathologies similar toenteropathy, such as experienced in disorders such as colitis,inflammatory bowel disease (IBD), psoriasis, rheumatoid arthritis,multiple sclerosis, and the like. It is known that several signalingtarget that have a role in enteropathy, such as stat3, akt, and mTor,also play a role in such other inflammatory diseases. Accordingly, suchother diseases and conditions are encompassed by the present disclosure.

In this aspect, the method comprises administering an effective amountof a TDMM, or TDMM precursor, to the subject, as described above. Asdescribed, in some embodiments, at least some components of themicrobiota are induced to produce higher levels of the TDMM.

In some embodiments, administration of the NSAID to the subject hascaused or is suspected of being capable of causing enteropathy in thesubject. In some embodiments, the NSAID has caused or is suspected ofcausing the relative decrease of a gram-positive component in themicrobiota, and/or conversely the NSAID has caused or is suspected ofcausing the relative increase of a gram-negative component of themicrobiota.

Representative NSAIDs and TDMMs, and their administrations, aredescribed above.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising at least one TDMM, or precursor or prodrug thereof, asdescribed above. If derived from microbiota or a bacterial source, theTDMM is preferably isolated or substantially isolated from the bacterialsource. In some embodiments, the pharmaceutical composition alsocomprises at least one NSAID composition, as described above.Accordingly, the disclosure provides for a pharmaceutical compositionwith co-formulation of an NSAID and a TDMM appropriate for a single,effective dose in a subject that reduces the chance or severity ofenteropathy in the subject due to the NSAID.

As the present disclosure has established that administration of a TDMMhas ameliorative and/or therapeutic effects for inflammation of the gut,for example, as caused by NSAID administration, the disclosure alsoprovides a method of treating a condition characterized by inflammationin the GI tract. The method comprises administering an effective amountof a tryptophan derived microbiota metabolite (TDMM), or a precursor,prodrug, or acceptable salt thereof, to a subject in need thereof. Theinflammation can associated with administration of an NSAID to thesubject. Alternatively, the inflammatory condition can have a similaronset or presentation as NSAID induced inflammation in the GI tract.

The pharmaceutical composition can be formulated appropriately usingroutine and conventional aspects of the art for appropriate routes ofadministration. Exemplary routes of administration appropriate for thecontemplated applications include intra peritoneal (IP), intravenous(IV), topical, parenteral, intradermal, transdermal, oral (e.g., vialiquid or pill), and respiratory (e.g., intranasal mist) routes.

The disclosed compositions are preferably pharmaceutically acceptableand can contain pharmaceutically acceptable carriers, concentrations ofsalt, buffering agents, preservatives, other immune modulators, andoptionally other therapeutic ingredients. The term“pharmaceutically-acceptable carrier” as used herein, means one or morecompatible solid or liquid filler, dilutants or encapsulating substanceswhich are suitable for administration to a human or other animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being comingled with active agents of the presentinvention, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

Unless specifically defined herein, all terms used herein have the samemeaning as they would to one skilled in the art of the presentinvention. Practitioners are particularly directed to Sambrook J., etal. (eds.) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Press, Plainsview, N.Y. (2001); Ausubel F. M., et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, New York(2010); and Coligan J. E., et al. (eds.), Current Protocols inImmunology, John Wiley & Sons, New York (2010) for definitions and termsof art.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike, are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to indicate, in the sense of“including, but not limited to.” Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below,” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. It is understoodthat, when combinations, subsets, interactions, groups, etc., of thesematerials are disclosed, each of various individual and collectivecombinations is specifically contemplated, even though specificreference to each and every single combination and permutation of thesecompounds may not be explicitly disclosed. This concept applies to allaspects of this disclosure including, but not limited to, steps in thedescribed methods. Thus, specific elements of any foregoing embodimentscan be combined or substituted for elements in other embodiments. Forexample, if there are a variety of additional steps that can beperformed, it is understood that each of these additional steps can beperformed with any specific method steps or combination of method stepsof the disclosed methods, and that each such combination or subset ofcombinations is specifically contemplated and should be considereddisclosed. Additionally, it is understood that the embodiments describedherein can be implemented using any suitable material such as thosedescribed elsewhere herein or as known in the art.

Publications cited herein and the subject matter for which they arecited are hereby specifically incorporated by reference in theirentireties.

The following describes a study demonstrating that microbiota-derivedmetabolite indole decreases mucosal inflammation and injury in a murinemodel of NSAID enteropathy.

Brief Summary

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the mostfrequently used classes of medications in the world. Unfortunately,NSAIDs induce an enteropathy associated with high morbidity andmortality. Although the pathophysiology of this condition involves theinteraction of the gut epithelium, microbiota, and NSAIDs, the precisemechanisms by which microbiota influence NSAID enteropathy are unclear.It was proposed that a possible mechanism is that the microbiota mayattenuate the severity of disease by specific metabolite-mediatedregulation of host inflammation and injury. The microbiota-derivedtryptophan-metabolite indole is abundant in the healthy mammalian gutand otherwise positively influences intestinal health. Thus the effectsof indole administration on NSAID enteropathy were examined.

Mice (n=5 per group) were treated once daily for 7 days with an NSAID(indomethacin; 5 mg/kg), indole (20 mg/kg), indomethacin plus indole, orvehicle only (control). Outcomes compared among groups included:microscopic pathology; fecal calprotectin concentration; proportion ofneutrophils in the spleen and mesenteric lymph nodes; fecal microbiotacomposition and diversity; small intestinal mucosal transcriptome; and,fecal tryptophan metabolites.

It was observed that co-administration of indole with indomethacinsignificantly reduced mucosal pathology scores, fecal calprotectinconcentrations, and neutrophilic infiltration of the spleen andmesenteric lymph nodes induced by indomethacin. Additionally,co-administration of indole with indomethacin modulated NSAID-inducedperturbation of the microbiota, fecal metabolites, and inferredmetagenome. Finally, co-administration of indole with indomethacinabrogated a pro-inflammatory gene expression profile in the smallintestinal mucosa induced by indomethacin.

These data demonstrate that the microbiota-derived metabolite indoleattenuated multiple deleterious effects of NSAID enteropathy, includingmodulating inflammation mediated by innate immune responses and alteringindomethacin-induced shift of the microbiota.

Introduction

As described above, it was hypothesized that aspects of a healthymicrobiota can prevent or ameliorate the negative side effects of NSAIDadministration, such as enteropathy. A mechanism by which the microbiotamight influence NSAID-induced intestinal mucosal damage is by producingmetabolites that protect intestinal epithelial cells. Previously, theinventors identified tryptophan metabolites, including indole, as animportant class of GI microbiota-derived compounds. Indole is aquorum-sensing molecule produced by bacterial metabolism of L-tryptophanthat mediates communication among bacterial population and inter-kingdomsignaling between the host and microbe. Indole improves barrier functionand decreases intestinal inflammation in vitro and in vivo. Moreover,several other tryptophan metabolites reportedly exert similar salutaryeffects on the intestinal epithelium. The inventors investigated whetherindole might mitigate the severity of NSAID enteropathy. Toward thisend, indole was co-administered with the NSAID indomethacin anddemonstrated a reduction in severity of mucosal injury caused byindomethacin alone. To determine whether the protective effects ofindole were associated with alterations in the GI microbiota, theeffects of administration of NSAIDs, indole, and their co-administrationon the composition and diversity of the fecal microbiota werecharacterized. It was observed that the co-administration of indole withindomethacin resulted in maintenance of or even an increase of animportant member of the Firmicutes phylum. Finally, to better understandthe mechanisms of NSAID enteropathy and the effects of indole on theseprocesses, RNA sequencing (RNA-Seq) was performed on the distal smallintestinal mucosa in mice that were untreated or treated withindomethacin, indole alone, or the combination of indomethacin andindole. Pro-inflammatory pathways associated with innate immuneresponses were up-regulated by indomethacin administration relative tocontrol mice, and co-administration of indole significantly mitigatedthe up-regulation of these pathways concomitant with reduced GIpathology.

Results Indole Reduced the Severity of NSAID Enteropathy

Calprotectin enzyme-linked immunosorbent assay (ELISA) of fecal samplescollected on days 0 and 6 revealed that co-administration of indole withindomethacin significantly decreased fecal calprotectin levels (FIG.1A). Moreover, attenuation of NSAID-induced small intestinal damage wasconfirmed by microscopic pathology scores (FIGS. 1B and 1C) of mice inwhich indole was co-administered with indomethacin. The reduction inmean microscopic pathology scores were corroborated by morphologicalparameters which revealed maintenance of villus height:crypt depth ratio(FIG. 1D) and thickness of the submucosal layers (FIGS. 6A and 6B) inmice in which indole was co-administered. Indomethacin had a negligibleeffect on the large intestine (data not shown), consistent with evidencethat indomethacin induces small intestinal ulcerations in mice in alocation similar to where NSAIDs injure people.

Indole Reduced Indomethacin-Induced Neutrophilic Infiltration of Spleenand Mesenteric Lymph Node (MLN)

Neutrophilic inflammation is primarily responsible for NSAIDenteropathy, therefore, we quantified the abundance of neutrophils inthe spleen and MLNs as measures of systemic neutrophilic response andtrafficking of neutrophils though the GI tract, respectively.²⁹⁻³⁰Indomethacin treatment resulted in a significant increase in neutrophils(defined as both CD11b- and GR-1 double-positive; FIG. 2) in both thespleen (FIG. 1E) and MLNs (FIG. 1F), and co-administration of indolesignificantly decreased neutrophilic infiltration of these tissues.Taken together, these data demonstrate that indomethacin induced smallintestinal injury in mice that was accompanied by neutrophilicinfiltration of the spleen and mesenteric lymph node and thatco-administration of indole attenuated the small intestinal injury andlimited neutrophilic infiltration of the spleen and mesenteric lymphnodes caused by indomethacin.

Indole Prevented Indomethacin-Induced Fecal Microbiota Shift andAlterations of the Inferred Metagenome

Because NSAIDs alter the intestinal microbiota, we evaluated thecomposition and diversity of the fecal microbiota in the four groups ofmice. To adjust for uneven sequencing depth among the samples, eachsample was rarefied to an even sequencing depth of 10,000 reads persample prior to analysis. Alpha rarefaction curves and Good's coverageindex estimates indicated that over 90% of the species were representedacross all samples at this sequencing depth (FIGS. 8A-8D). Usinganalysis of similarities (ANOSIM), no significant difference in theunweighted Unifrac distance metric among the groups was observed on day0 (R=0.10; P=0.11); however, by day 7 there was a significant differenceamong the groups (R=0.3007; P=0.0031). Pairwise comparisons betweengroups revealed that a significant difference in the Unifrac distancemetric existed only between NSAID and control animals (TABLE 2) and thatco-administration of indole with indomethacin attenuated this change inthe beta diversity of the fecal microbiota.

TABLE 2 Pairwise analysis of similarity (ANOSIM) R-values based on theunweighted Unifrac distance metric at day 7. Only the difference betweencontrol and NSAIDs was significantly different than 0. Pairwise AOSIMR-values Control NSAID Indole NSAID + Indole Control 0.5487* 0.2 0.2813NSAID 0.5487* 0.2615 0.4444 Indole 0.2 0.2615 0.1938 NSAID + Indole0.2813 0.4444 0.1938 *R value is significantly (P < 0.05) different thana value of 0.

The primary gram-positive and gram-negative phyla found in murine fecesare Firmicutes and Bacteroidetes, respectively. At the phylum level,principal component analysis (PCA) revealed a separation ofNSAID-treated mice from the other groups characterized by increase inmembers of the phyla Bacteroidetes in the NSAID-treated animals betweenday 0 and day 7 (FIGS. 2A and 2B). Interestingly, PCA revealedqualitatively that co-administration of indole counteracted the increasein Bacteroidetes and instead appeared to shift the group closer to thephylum Firmicutes. Based on the results of PCA, we compared theabundance of members of the phyla Firmicutes and Bacteroidetes and foundno significant difference after treatment with NSAIDs, indole, or theircombination (FIGS. 9A and 9B). At lower taxonomic levels, however,significant differences were observed. For example, co-administration ofindole with indomethacin prevented a decrease in Clostridiales andinstead this group had a significant increase in several members of theClostridales order (FIG. 2C). Furthermore, similarity percentage(SIMPER) based on the Bray Curtis dissimilarity measure at phylum,order, and family levels further confirmed that indomethacin treatmentresulted in the gain of members of the Bacteroidales S24-7 family, amajor family of Bacteroidetes found in murine feces, indicating thatgain of this family contributed to the dissimilarity between NSAID andthe other groups.³³ Co-administration of indole prevented this increasein Bacteroidales and instead resulted in an increase in Clostridialeswith marginal increases in several other members of the Firmicutes phyla(TABLES 3A-5 and FIG. 2D).

PCA of the inferred metagenome revealed clustering and separation of theNSAID-treated mice from the other groups (FIGS. 2E and 2F), althoughanalysis of similarity (ANOSIM) based on the Bray Curtis dissimilaritymetric indicated this difference was not significant (P>0.05).Consistent with the microbiota data, the distance among groups wasgreatest between the NSAID group and the NSAID+indole group. The majorup- and down-regulated inferred functional pathways between NSAID andNSAID+indole groups were tabulated (TABLE 6).

TABLES 3A-3C disclose the results of a pair-wise similarity percentageanalysis (SIMPER) analysis based on the bray Curtis dissimilaritymeasure of fecal 16S rRNA data at the phylum level of each treatmentgroup compared to the control group based on day 7 feces.TABLE 3A: The first column identifies the bacterial phylum explained bythat row, the second column shows % contribution of that phylum to theBray Curtis dissimilarity measure between the 2 groups, the third columntallies the cumulative Bray Curtis dissimilarity measure thus farrepresented in the table, and the last 2 columns show mean abundance incontrol mice and mean abundance for NSAID-treated mice;TABLE 3B: Control mice and NSAID+indole-treated mice, andTABLE 3C: control mice and indole-treated mice.

A) % Contribution % Cumulative Control mean NSAID mean Phylum todissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Firmicutes 49.22 49.22 32.2 17.1 k_Bacteria; p_Bacteroidetes 47.3296.54 67.1 81.2 k_Bacteria; p_Tenericutes 3.215 99.75 0.611 1.66k_Bacteria; p_Actinobacteria 0.1128 99.87 0.0552 0.0476 k_Bacteria;p_Proteobacteria 0.05056 99.92 0.0171 0.00317 Unassigned; Other 0.0466899.96 0.0171 0.00317 k_Bacteria; p_Cyanobacteria 0.01555 99.98 0.00380.00634 k_Bacteria; p_Verrucomicrobia 0.01167 99.99 0.0038 0 k_Bacteria;p_Acidobacteria 0.009721 100 0 0.00317 k_Bacteria; Other 0 100 0 0

NSAID + Indole B) % Contribution % Cumulative Control mean mean Phylumto dissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Firmicutes 49.41 49.41 32.2 44.5 k_Bacteria; p_Bacteroidetes 49.1998.6 67.1 54.7 k_Bacteria; p_Tenericutes 1.174 99.77 0.611 0.671k_Bacteria; p_Actinobacteria 0.09981 99.87 0.0552 0.0452 k_Bacteria;p_Proteobacteria 0.06521 99.94 0.0171 0.0262 Unassigned; Other 0.0319499.97 0.0171 0.0143 k_Bacteria; p_Cyanobacteria 0.01197 99.98 0.00380.00238 k_Bacteria; p_Verrucomicrobia 0.01197 99.99 0.0038 0.00238k_Bacteria; Other 0.006652 100 0 0.00238 k_Bacteria; p_Acidobacteria 0100 0 0

C) % Contribution to % Cumulative Control mean Indole mean Phylumdissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Bacteroidetes 49.01 98.22 32.2 24.6 k_Bacteria; p_Firmicutes 49.2149.21 67.1 74.7 k_Bacteria; p_Tenericutes 1.37 99.59 0.611 0.656k_Bacteria; p_Actinobacteria 0.1894 99.78 0.0552 0.0381 k_Bacteria;p_Proteobacteria 0.09238 99.87 0.0171 0.0285 Unassigned; Other 0.0646799.94 0.0171 1.90E−03 k_Bacteria; p_Cyanobacteria 0.02925 99.97 3.80E−039.51E−03 k_Bacteria; p_Verrucomicrobia 0.0169 99.98 3.80E−03 1.90E−03k_Bacteria; Other 0.0154 100 0 3.81E−03 k_Bacteria; p_Acidobacteria 0100 0 0TABLES 4A and 4B disclose the results of a pair-wise SIMPER analysisbased on the Bray Curtis dissimilarity measure of fecal 16S rRNA data atthe order level of NSAID and NSAID+indole groups compared to the controlgroup based on day 7 feces.TABLE 4A: The first column identifies the bacterial order explained bythat row, the second column shows % contribution of that order to theBray Curtis dissimilarity measure between the two groups, the thirdcolumn tallies the cumulative Bray Curtis dissimilarity measure thus farrepresented in the table, and the last two columns show mean abundancein control mice and mean abundance for NSAID-treated mice; andTABLE 4B: Control mice and NSAID+indole-treated mice.

A) % Contribution % Cumulative Control mean NSAID mean Order todissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales 45.9 45.9 67.1 81.2k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales 32.46 78.36 25.114.2 k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales 12.33 90.695.2 2.65 k_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales 4.94895.64 1.7 0.0412 k_Bacteria; p_Tenericutes; c_Mollicutes; o_RF39 3.27398.91 0.523 1.62 k_Bacteria; p_Firmicutes: c_Erysipelotrichi; 0.464199.38 0.211 0.136 o_Erysipelotrichales k_Bacteria; p_Tenericutes;c_Mollicutes; 0.2339 99.61 0.0875 0.0317 o_Anaeroplasmatales k_Bacteria;p_Actinobacteria; c_Coriobacteriia; 0.1056 99.72 0.0533 0.0381o_Coriobacteriales k_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales0.06603 99.78 0.0228 0.0412 Unassigned; Other; Other; Other 0.0452899.83 0.0171 0.00317 k_Bacteria; p_Proteobacteria;c_Alphaproteobacteria; 0.0283 99.85 0.00951 0 o_Rickettsialesk_Bacteria; p_Actinobacteria; c_Actinobacteria; 0.02641 99.88 0.00190.00951 o_Actinomycetales k_Bacteria; p_Bacteroidetes; c_Cytophagia;0.01132 99.89 0.00381 0 o_Cytophagales k_Bacteria; p_Firmicutes;c_Bacilli; Other 0.01132 99.9 0.00381 0

NSAID + indole B) % Contribution % Cumulative Control mean mean Order todissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales 46.65 46.65 25.1 41.8k_Bacteria; p_Bacteroidetes; c_Bacteroidia; 38.49 85.13 67.1 54.7o_Bacteroidales k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales9.715 94.85 5.2 1.33 k_Bacteria; p_Firmicutes; c_Bacilli;o_Turicibacterales 3.667 98.52 1.7 1.28 k_Bacteria; p_Tenericutes;c_Mollicutes; o_RF39 0.8235 99.34 0.523 0.587 k_Bacteria; p_Firmicutes;c_Erysipelotrichi; 0.2041 99.54 0.211 0.119 o_Erysipelotrichalesk_Bacteria; p_Tenericutes; c_Mollicutes; 0.1822 99.73 0.0875 0.0832o_Anaeroplasmatales k_Bacteria; p_Actinobacteria; c_Coriobacteriia;0.07184 99.8 0.0533 0.0404 o_Coriobacteriales k_Bacteria;p_Proteobacteria; c_Alphaproteobacteria; 0.04164 99.84 0.00951 0.019o_Rickettsiales k_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales0.03228 99.8 0.0228 0.0166 Unassigned; Other; Other; Other 0.02499 99.90.0171 0.0143

TABLES 5A and 5B disclose the results of a pair-wise SIMPER analysisbased on the Bray Curtis dissimilarity measure of fecal 16S rRNA data atthe family level of NSAID and NSAID+indole groups compared to thecontrol group based on day 7 feces. TABLE 5A: The first columnidentifies the bacterial family explained by that row, the second columnshows % contribution of that family to the Bray Curtis dissimilaritymeasure between the 2 groups, the third column tallies the cumulativeBray Curtis dissimilarity measure thus far represented in the table, andthe last 2 columns show mean abundance in control mice and meanabundance for NSAID-treated mice; and

TABLE 5B: Control mice and NSAID+indole-treated mice.

A) % Contribution % Cumulative Control mean NSAID mean Family todissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 43.49 43.49 67.1 81.2f_S24-7 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 16.760.19 7.24 1.31 f_Lachnosoiraceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; 11.57 71.77 5.19 2.48 f_Lactobacillaceae k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_ 11.12 82.88 11 7.67k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 5.042 87.935.04 3.53 f_Ruminococcaceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Turicibacterales; 4.688 92.61 1.7 0.0412 f_Turicibacteraceaek_Bacteria; p_Tenericutes; c_Mollicutes; o_RF 39; f_ 3.101 95.72 0.5231.62 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 2.45298.17 1.7 1.68 f_Clostridiaceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; 0.4826 98.65 0.00381 0.171 f_Enterococcaceaek_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales;0.4397 99.09 0.211 0.136 f_Erysipelotrichaceae k_Bacteria;p_Tenericutes; c_Mollicutes; o_Anaeroplasmatales; 0.2216 99.31 0.08750.0317 f_Anaeroplasmataceae k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.1108 99.42 0.0457 0.00634 f_Dehalobacteriaceaek_Bacteria; p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales;0.1001 99.52 0.0533 0.0381 f_Coriobacteriaceae k_Bacteria; p_Firmicutes;c_Bacilli; o_Bacillales; 0.05005 99.57 0.0038 0.019 f_Staphylococcaceaek_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales; Other 0.05004 99.620.00381 0.019 Unassigned; Other; Other; Other; Other 0.0429 99.67 0.01710.00317 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; Other0.0429 99.71 0.0171 0.00317 k_Bacteria; p_Firmicutes; c_Bacilli;o_Bacillales; 0.03217 99.74 0.0133 0.00317 f_Bacillaceae k_Bacteria;p_Proteobacteria; c_Alphaproteobacteria; 0.02681 99.77 0.00951 0o_Rickettsiales; f_mitochondria k_Bacteria; p_Actinobacteria;c_Actinobacteria; 0.02502 99.79 0.0019 0.00951 o_Actinomycetales;f_Micrococcaceae k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.02145 99.81 0.00381 0.00634 f_[Mogibacteriaceae]k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; 0.01608 99.830.00571 0 f_Streptococcaceae k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidale; 0.01608 99.85 0.0019 0.00634 f_Bacteroidaceaek_Bacteria; p_Firmacutes; c_Bacilli; o_Lactobacillales; 0.01608 99.860.00571 0 f_Aerococcaceae k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.01429 99.88 0.00571 0.00317 f_Prevotellaceaek_Bacteria; p_Bacteroidetes; c_Cytophagla; o_Cytophagales; 0.01072 99.890.00381 0 f_Cytophagacae k_Bacteria; p_Firmacutes; c_Bacilli; Other;Other 0.01072 99.9 0.00381 0

NSAID + indole B) % Contribution % Cumulative Control mean mean Familyto dissimilarity dissimilarity % abundance % abundance k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 34.58 34.58 67.1 54.7f_S24-7 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; f_22.57 57.15 11 19.4 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 13.69 70.83 5.04 9.99 f_Ruminococcaceae k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 10.45 81.29 7.24 9.12f_Lachnosoiraceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; 8.725 90.01 5.19 1.31 f_ Lactobacillaceae k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 4.964 94.98 1.7 3.05f_Clostridiaceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Turicibacterales; 3.296 98.27 1.7 1.28 f_Turicibacteraceae k_Bacteria;p_Tenericutes; c_Mollicutes; o_RF 39; f_ 0.7401 99.01 0.523 0.587k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales;0.1834 99.2 0.211 0.119 f_Erysipelotrichaceae k_Bacteria; p_Tenericutes;c_Mollicutes; o_Anaeroplasmatales; 0.1637 99.36 0.0875 0.0832f_Anaeroplasmataceae k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; Other 0.1628 99.52 0.0171 0.0999 k_Bacteria;p_—Firmicutes; c_Clostridia; o_Clostridiales; 0.09731 99.62 0.04570.0571 f_Dehalobacteriaceae k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales; 0.06456 99.68 0.0533 0.0404f_Coriobacterlaceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; 0.03743 99.72 0.00381 0.019 f_Enterococcaceaek_Bacteria; p_Proteobacteria; c_Alphaproteobacteria; 0.03743 99.760.00951 0.019 o_Rickettsiales; f_mitochondria Unassigned; Other; Other;Other; Other 0.02246 99.78 0.0171 0.0143 k_Bacteria; p_Firmicutes;c_Bacilli; o_Bacillales; 0.02058 99.8 0.0133 0.00475 f_Bacillaceaek_Bacteria; p_Firmicutes; c_Bacilli; o_Bacillales; Other 0.01871 99.820.00381 0.00951 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.01403 99.83 0.0019 0.00713 f_Peptostreptococcaceae k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 0.01123 99.85 0.0019 0.00476o_Aeromonadales; f_Succinivibrionaceae k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.01122 99.86 0.00571 0 f_Prevotellaceaek_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales; 0.01122 99.870.00571 0 f_Streptococcaceae k_Bacteria; p_Firmacutes; c_Bacilli;o_Lactobacillales; 0.01122 99.88 0.00571 0 f_Aerococcaceae k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.01029 99.89 0.003810.00238 f_[Mogibacteriacaea] k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.009357 99.9 0 0.00476 f_[Tissierellaeae]

NSAID NSAID + NSAID + Control NSAIDmean vs. indole indole vs. %Contribution to mean % % Control mean % Control Taxon dissimilarityabundance abundance Direction abundance Direction EnvironmentalInformation 13.88 5.29% 3.82% ↓ 6.35% ↑ Processing; Membrane Transport;Transporters Environmental Information 6.132 2.36% 1.71% ↓ 2.76% ↑Processing; Membrane Transport; ABC transporters Genetic InformationProcessing; 3.171 1.14% 0.31% ↓ 1.38% ↑ Transcription; Transcriptionfactors Cellular Processes; Cell Motility; 2.895 0.61% 0.81% ↑ 0.93% ↑Bacterial motility proteins Genetic Information Processing; 2.536 2.70%2.94% ↑ 2.45% ↓ Translation; Ribosome Unclassified; Cellular Processesand 1.971 0.40% 1.00% ↑ 0.55% ↑ Signalling; Sporulation Metabolism;Energy Metabolism; 1.944 1.46% 1.65% ↑ 1.28% ↓ Oxidative phosphorylationEnvironmental Information 1.746 1.19% 0.20% ↓ 1.37% ↑ Processing; SignalTransduction; Two-component system Unclassified; Cellular Processes and1.578 0.75% 0.17% ↓ 0.62% ↓ Signalling; Membrane and intracellularstructural molecules Genetic Information Processing; 1.572 2.98% 3.12% ↑2.81% ↓ Replication and Repair DNA repair and recombination proteinsCellular Processes; Cell Motility; 1.5 0.33% 0.91% ↑ 0.50% ↑ Flagellarassembly Environmental Information 1.486 0.27% 0.18% ↓ 0.22% ↓Processing; Membrane Transport; Phosphotransferase system (PTS)Metabolism; Nucleotide 1.47 2.11% 2.24% ↑ 1.97% ↓ Metabolism; Pyrimidinemetabolism Metabolism; Energy Metabolism; 1.327 1.18% 0.40% ↓ 1.06% ↓Carbon fixation pathways in prokaryotes Cellular Processes; CellMotility; 1.322 0.31% 1.30% ↑ 0.46% ↑ Bacterial chemotaxis Metabolism;Metabolism of 1.24 0.53% 2.42% ↑ 0.66% ↑ Cofactors and Vitamins;Porphyrin and chlorophyll metabolism Metabolism; Nucleotide 1.191 2.31%0.90% ↓ 2.17% ↓ Metabolism; Purine metabolism Unclassified; Metabolism;Energy 1.178 1.14% 1.25% ↑ 1.04% ↓ metabolism Metabolism; Carbohydrate1.162 0.79% 1.25% ↑ 0.68% ↓ Metabolism; Citrate cycle (TCA cycle)Genetic Information Processing; 1.159 1.15% 0.61% ↓ 1.05% ↓ Folding,Sorting and Degradation; Chaperones and folding catalysts Metabolism;Glycan Biosynthesis 1.114 0.50% 1.48% ↑ 0.42% ↓ and Metabolism;Lipopolysaccharide biosynthesis proteins Genetic Information Processing;1.078 1.39% 0.50% ↓ 1.29% ↓ Replication and Repair; DNA replicationproteins Metabolism; Glycan Biosynthesis 1.073 0.40% 1.55% ↑ 0.32% ↓ andMetabolism; Lipopolysaccharide biosynthesis Metabolism; Amino Acid0.9791 1.18% 1.14% ↓ 1.10% ↓ Metabolism; Alanine, aspartate andglutamate metabolism Metabolism; Glycan Biosynthesis 0.9663 0.64% 1.27%↑ 0.60% ↓ and Metabolism; Other glycan degradation Genetic InformationProcessing; 0.9311 1.05% 2.12% ↑ 0.97% ↓ Replication and Repair;Homologous recombination Metabolism; Amino Acid 0.9101 1.57% 1.65% ↑1.49% ↓ Metabolism; Amino Acid related enzymes Genetic InformationProcessing; 0.8961 1.47% 0.70% ↓ 1.37% ↓ Translation Ribosome BiogenesisUnclassified; Cellular Processes and 0.8215 0.41% 3.54% ↑ 0.34% ↓Signaling; Pores ion channels Metabolism; Metabolism of 0.7986 0.77%0.49% ↓ 0.71% ↓ Cofactors and Vitamins; One carbon pool by folateUnclassified; Metabolism; Others 0.7909 0.79% 0.84% ↑ 0.85% ↑Metabolism; Metabolism of 0.77 0.47% 1.66% ↑ 0.41% ↓ Cofactors andVitamins; Folate biosynthesisTABLE 6 discloses the results of a pair-wise SIMPER analysis based onthe Bray Curtis dissimilarity measure of the inferred metagenome of thefecal microbiota of NSAID and NSAID+indole groups compared to thecontrol group based on day 7 feces. The first column identifies the KEGGpathway explained by that row, the second column shows % contribution ofthat pathway to the Bray Curtis dissimilarity measure between the 2groups, the third column tallies the cumulative Bray Curtisdissimilarity measure thus far represented in the table, and the last 4columns show mean abundance in control mice and mean abundance forNSAID-treated mice, direction of change for that comparison,NSAID+indole-treated mice and directional change for that comparisonversus control mice.

Co-Administration of Indole Prevents the Indomethacin-InducedTryptophan-Derived Metabolite Disruption in Feces

Multiple tryptophan-derived metabolites produced by the microbiotapredicted to be bioactive and exert effects on the host have beenidentified (Sridharan G V, Choi K, Klemashevich C, Wu C, Prabakaran D,Pan L B, Steinmeyer S, Mueller C, Yousofshahi M, Alaniz R C, et al.Prediction and quantification of bioactive microbiota metabolites in themouse gut. Nature Communications 2014; 5:5492, incorporated herein byreference in its entirety). Given the importance of the microbiota inNSAID enteropathy, the effects of indole on the intestinal epitheliumand microbiota were examined (FIGS. 2A-2F) and were correlated withtryptophan metabolites. No single tryptophan metabolite wassignificantly correlated with fecal calprotectin (data not shown). Onlythe metabolite tyramine was significantly (P<0.01) correlated withmicroscopic pathology scores (TABLE 7). Examination of the fecal profileof all tryptophan metabolites using PCA revealed a visible separation ofthe NSAID-treated mice from the remaining groups on day 7, whileco-administration of indole appeared to prevent this separation (TABLE7); however, ANOSIM of the Bray Curtis dissimilarity measure indicatedthis apparent difference was not significant (P=0.55). Examination oftryptophan metabolite concentrations by treatment group revealed asignificant increase in tyramine in the NSAID group that was attenuatedby co-administration of indole (TABLE 2); moreover, several othertryptophan metabolites tended to be increased only in the feces of micetreated exclusively with indomethacin, indicating that NSAID treatmentcaused differences that were attenuated by co-administration of indole(TABLE 8). Relative to controls, mice in the indole-only andindomethacin-only groups had significantly more indole; although fecalindole concentrations were generally higher in the NSAID+indole mice,this difference was not significant.

TABLE 7 discloses a correlation of fecal tryptophan metabolites withhistopathology scores≤2 or>2. Only the tryptophan metabolite tyraminecorrelated with microscopic pathology score.

Metabolite Score < 2 Median (Range) Score > 2 Median (Range) P valueHydroxytryptophan 11.9 (3.5-24.5) 27.3 (6.0-28.4) 0.3097 Hydroxyindole23.7 (15.7-58.1) 38.3 (18.0-78.6) 0.3301 Arginine 11.2 (1.9-241.6) 8.9(4.9-24.6) 1 Glutamic Acid 404 (56-4,857) 345 (77-1,814) 0.953Indole-3-acetamide 0.001 (<0.001-0.004) 0.001 (<0.001-0.002) 0.5941Indole-3-actetate 0.021 (0-0.080) 0 (0-0.238) 0.3879Indole-3-carboxaldehyde 0.324 (0.162-0.867) 0.441 (0.179-0.582) 0.5135Indole 21.5 (0-157.2) 97.1 (53.3-131.3) 0.0922 Kyneurine 0 (0-0.184) 0(0 to 0.061) 0.6287 Ornithine 6.2 (2.0-9.6) 13.3 (6.3-32.8) 0.3277Phenylalanine 2053 (1109-6247) 3,108 (1,192-3,957) 0.8591 Serotonin 1.28(0.62-1.73) 2.60 (0.60-3.32) 0.0992 Tryptamine 0.076 (0.050-0.314) 0.094(0.053-0.166) 0.2065 Tryptophan 3.38 (1.33-19.22) 4.66 (1.64-7.95)0.5135 Tyramine 2.32 (1.44-11.66) 22.77 (2.68-208.17) 0.008 Tyrosine48.0 (20.9-231.0) 24.88 (10.52-134.37) 0.206

TABLE 8 Group effects of tryptophan-derived metabolites identifying thedirection and magnitude of change in fecal concentration between Days 0and 7. Several tryptophan-derived metabolites tended to be increased inthe NSAID treated mice (5-hydroxytryptamine, 5-hydroxyindole, indole,serotonin, tyramine). Fecal concentrations of indole increased in thegroups in which indole was administered. Metabolite Group effectMagnitude (95% CI) P value 5-Hydroxytryptamine ↑ NSAID only 13.9 (−1.4to 29.3) 0.1029 5-Hydroxyindole ↑ NSAID only 27.2 (−2.2 to 56.6) 0.0657Arginine ↔ Glutamine ↔ Indole-3-acetamide ↔ Indole-3-acetate ↔Indole-3-carboxaldehyde ↔ Indole ↑ NSAID 6.7 (1.6 to 28.2) 0.0255↑Indole 5.3 (1.1 to 24.7) 0.0364 ↑NSAID + Indole 4.7 (0.9 to 24.7)0.0641 Kyneurine ↔ Ornithine ↔ Phenylalanine ↔ Serotonin ↑ NSAID only1.6 (−0.3 to 3.5) 0.0895 Tryptamine ↔ Tryptophan ↔ Tyramine ↑ NSAID only6.7 (1.3 to 33.6) 0.0024 Tyrosine ↔

Co-Administration of Indole Attenuates NSAID-Induced Pro-InflammatoryMucosal Transcriptomic Changes

RNA sequencing (RNA-Seq) of the distal small intestinal mucosa wasperformed to examine the in vivo transcriptomic changes associated withNSAID enteropathy and to gain insight into how the co-administration ofindole altered gene expression. The top genes that were significantlyup- or down-regulated (fold change≥2) in NSAID-treated mice relative tocontrol mice, indole-treated mice relative to control mice, andNSAID+indole-treated mice relative to control mice were tabulated. TheIngenuity Pathway Analysis software package was used to identifypathways represented by differentially expressed genes. Severalcanonical pathways were altered in NSAID-treated mice relative tocontrols (FIG. 3A). Several of the pathways that were modulated by NSAIDadministration were either shifted to the opposite direction (i.e.,inhibited or activated, respectively), or the degree of activation orinhibition was markedly attenuated by co-administration of indole (FIG.3B).

Moreover, transcription of specific pro-inflammatory cytokines(interleukin [IL]-1α, IL-1β, tumor necrosis factor (TNF), IL-6) andchemokines (chemokine C-X-C motif [CXC]L1, CXCL3, CXCL2, CXCL5, CCL2,CCL7) was significantly up-regulated in NSAID-treated mice; however,when indole was co-administered the degree of up-regulation was notsignificantly different than control mice (FIGS. 10A and 10B). Based onour results, a proposed schema of the interaction of NSAIDs, the hostmucosal epithelium, the microbiota, and indole is presented (FIG. 4).

It was also observed that the exfoliated intestinal epithelial cells(IEC) and tissue-level gene expression profiles were similar (notshown). Thus, the differences in the exfoliated IEC transcriptomebetween control versus indomethacin-treated animals and control versusindole+indomethacin-treated animals were examined. For this purpose, thetop 40 canonical pathways altered in exfoliated IECs were sorted basedon differences in Z-scores between the 2 treatments (FIG. 3C).Similarly, the top 50 upstream regulators altered in exfoliated IECs areshown in FIG. 3D. While these data reveal pathways consistent withreduced inflammation, it is unclear how this anti-inflammatory effectoccurred and which of these upstream regulators mediate indomethacininduced GI injury and the protective effects observed by theco-administration of indole. To address this question, the network ofthe top 15 upstream regulators identified by our novel data reductionapproach was mapped and analyzed with 2-feature LDA in the context ofNSAID vs control animals (FIG. 5A). The fold change and false discoveryrate (FDR) P-values of these same molecules from the analysis of controlwas overlaid versus NSAID+indole (FIG. 5B), allowing identification ofthe molecules were altered (opposite fold-change either realized orpredicted). This resulted in the identification of 11 genes that werealtered between NSAID administration and NSAID+indole administration.

Discussion

Co-administration of indole attenuated small intestinal mucosal damageinduced by administration of indomethacin in mice as manifested byreduced microscopic pathology and fecal calprotectin concentration.Fecal calprotectin is a well-established, non-invasive indicator ofintestinal mucosal injury induced by NSAIDs in human patients and animalmodels, and correlates well with 4-day fecal excretion of¹¹¹Indium-labelled leukocytes. The findings of decreased fecalcalprotectin and decreased microscopic pathology have important clinicalimplications. A variety of NSAIDs are used widely for an array ofclinical conditions ranging from pain relief for minor injuries tomanagement of rheumatoid arthritis or cancer. The relatively low cost,high effectiveness, and lack of alternatives to NSAIDs indicate thattheir use will continue to be highly prevalent. Consequently, agentsthat might be co-administered with NSAIDs to diminish NSAID enteropathywould be clinically important. Further evaluation of indole toameliorate NSAID enteropathy in animal models and naturally occurringdisease is warranted by our findings.

Administration of NSAIDs increases the proportion of gram-negativeorganisms at the expense of gram-positive organisms in the intestinalmicrobiota, and this shift has been shown to contribute to NSAID-inducedintestinal injury. Specifically, NSAID administration decreases variousmembers of the class Clostridia and increases members of the classBacteroidia. Mice treated with indole and indomethacin did not have achange in the abundance of Bacteroidia but did have an increase inseveral members of the gram-positive family Clostridiales in concertwith diminution of the severity of intestinal mucosal damage. Evidenceexists that the microbiota plays an important role in the development ofNSAID enteropathy. Germ-free rats treated with NSAIDs develop lesssevere enteropathy than specific-pathogen-free rats or germ-free ratsthat have been colonized with gram-negative bacteria.³⁹ Toll-likereceptor (TLR) 4-deficient mice develop less severe lesions thanisogeneic TLR4-competent strains. Dramatic NSAID-induced alterations ofthe gut microbiota are well-documented and most often characterized by aloss of gram-positive bacteria with a concurrent increase ingram-negative bacteria. Moreover, this particular shift in themicrobiota has been associated with increased severity of intestinalmucosal injury, and preventing this shift can reduce mucosal injury. Theclassic indomethacin-induced increase in types of Bacteroidia weobserved in our study were not significant, but this was likelyattributable to limited power to detect a statistically significantdifference resulting from our small sample size. It is not clear whyincreased abundance of gram-negative bacteria worsens the severity ofNSAID enteropathy, but direct effects of LPS and the host innate immuneresponse to LPS appear to be important. It is also possible that loss ofbeneficial gram-positive bacteria is important. Commensal Clostridiahave been shown to be critically important in gut homeostasis,specifically members of Clostridium cluster XIVa and Clostridium clusterIV. Interestingly, several members of these two clostridial clusterswere increased in the NSAID-treated animals in which indole wasco-administered.

The host response to the microbiota might be more important than themicrobiota itself in the pathogenesis of NSAID enteropathy. Neutrophilsare key effector cells of innate immunity and are critically importantin the pathogenesis of NSAID enteropathy. Neutrophils are recruited tothe site of injury by the influx of luminal contents following increasedmucosal permeability. The resident innate immune cells present in theepithelium and lamina propria release cytokines and chemokines thatattract circulating neutrophils. These neutrophils, along with otherinnate immune cells, then release pro-inflammatory cytokines, typicallycharacterized by an abundance of the IL-1 super family, TNF-α, IL-6, andothers, that are responsible for the damage to the lower GI tract. Thiscritical role for neutrophils in NSAID enteropathy is supported by ourfindings of neutrophilic infiltration of both the spleen and MLNfollowing NSAID administration and reduced neutrophil concentrations inthese tissues with co-administration of indole. These data also affirmthe importance of the innate immune response in the pathophysiology ofNSAID enteropathy because many of the most up-regulated pathwaysidentified by RNA-Seq reflected the innate immune response (viz., NF-κBpathway, TLR signaling pathways, and the LPS/IL-1 response). Moreover,at the individual gene level, several of the classic pro-inflammatorycytokines associated with neutrophil activation and known to beimportant in NSAID enteropathy (e.g., TNF-α, IL-1, and IL-6) wereup-regulated among NSAID-treated mice. Co-administration of indole,however, attenuated or reversed up-regulation of genes associated withinnate immunity and inflammation that contribute to the pathogenesis ofNSAID enteropathy. In addition, several chemokines that attractneutrophils were up-regulated in the NSAID-treated mice and thisup-regulation was dramatically attenuated by the co-administration ofindole. These data indicate that indole can mitigate the host innateimmune response to the influx of luminal contents across injuredepithelia. Indole has been shown to inhibit nuclear factorkappa-light-chain-enhancer of activated B cells (NF-κB) signaling inintestinal epithelial cells. It is thus plausible that indole exertssimilar effects on neutrophils and other innate immune cells, therebyreducing cytokines dependent upon NF-κB signaling. Although NF-κBsignaling was activated by administration of both NSAID andNSAID+indole, activation of this pathway was greatly diminished byco-administration of indole with NSAIDs, suggesting indole mightmitigate NSAID damage in part by inhibiting NF-κB. It is important tonote that the mRNA for RNA-seq was isolated from mucosal scrapings.These scrapings likely contain epithelial cells as well as cellsresiding in the lamina propria (i.e., immune cells recruited to theinflamed gut due to loss of barrier function) and therefore we cannot besure the exact source of gene expression profiles. NSAID enteropathy ischaracterized as a mucosal injury and we felt examining thetranscriptome of this location would be more informative than examiningthe transcriptome of whole tissue. The observed anti-inflammatoryeffects of indole may be due to indole's effects on epithelial cells,immune cells in the lamina propria, or both.

It is unclear how indole prevented the characteristic shifts in thecomposition of the microbiota associated with NSAID administration.Indole has long been recognized as a quorum-sensing molecule so it ispossible that indole directly affected the microbial community by actingas an intra-kingdom signaling molecule. It is, however, also plausiblethat changes in the microbiota associated with NSAID use occur secondaryto mucosal inflammation because mucosal inflammation has been shown toalter the luminal environment. Indole might have attenuated mucosaldamage, which in turn prevented the expected changes in the microbiota.Finally, it has been shown that GI microbiota can vary with environmentincluding cage-dependent variation and cage-dependent clustering. Inorder to mitigate this phenomenon, after acclimation and immediatelyprior to starting the study (Day 0), mice were randomly assigned intotreatment groups and then were moved into cages based on the group towhich they were randomly assigned. There were no significant differencesin the fecal beta diversity among the groups at Day 0. It is possible,however, that were some cage-dependent microbiota changes that occurredover the 7 days of treatment that might have influenced the compositionof the microbiota after treatment.

Indole is present in the GI tract of humans and animals at relativelyhigh concentrations (˜250-1,100 μM). It has been speculated that,because intestinal epithelial cells are continually exposed to indole,indole may act as an interkingdom signaling molecule. Indeed theinventors have shown, in vitro, that indole does behave in this mannerand has anti-inflammatory effects on intestinal epithelial cells andupregulates expression of genes associated with tight cell junctions. Itwas tested, then, whether increasing the concentration of indole withinthe lumen of the GI tract might mitigate NSAID enteropathy because ofthe in vitro effects of indole on intestinal epithelial cells. Asexpected, mice gavaged with indole (alone or in combination withindomethacin) were observed to have increased fecal concentration ofindole. Interestingly, the NSAID-only treated mice also had increasedfecal concentrations of indole. Although both gram-negative andgram-positive bacteria produce indole, the list of gram-negativebacteria known to produce indole is much larger than gram-positivebacteria. Thus, the observed increase in gram-negative bacteria in theNSAID treated mice might explain their increased fecal concentrations ofindole. The beneficial effects of indole in this study were observed atthe distal small intestine, but the concentration of indole and themicrobiota diversity were determined using fecal samples. Fecalmetabolite and microbiota characterization do not always correlate wellwith those of more proximal mucosal locations in the GI tract.Therefore, it is possible that indole was present in higherconcentrations in the distal small intestine of mice treated with NSAIDcombined with indole compared with mice treated with the NSAID alone,thus contributing to the attenuation of NSAID-induced injury by indoleadministration. Interestingly, several tryptophan metabolites that actas neurotransmitters including serotonin and 5-hydroxytryptamine werealso increased in NSAID-treated mice. Hypermotility of the GI tract isinduced by NSAIDs and contributes to the pathophysiology of NSAIDenteropathy. The microbiota shift induced by NSAIDs might result inproduction of prokinetic metabolites that contribute to GIhypermotility.

In summary, indole supplementation of mice attenuates the deleteriouseffects of NSAIDs on the distal small intestine and modulatesNSAID-induced alterations in the composition of the fecal microbiota.The major events in the onset of NSAID enteropathy are intestinalepithelial cell death, increased mucosal permeability, influx of luminalcontents, and host innate immune response to the microbiota. Indolelikely reduces intestinal injury induced by NSAIDs at multiple levels,including neutrophilic infiltration, NSAID-induced dysbiosis, andpro-inflammatory pathways in the distal small intestine. Future workwill focus on interrogating the potential mechanisms by which indoleexerts this beneficial effect in order to further elucidate means tocontrol or prevent NSAID enteropathy.

Materials and Methods

Animal protocols were approved by the Texas A & M Institutional AnimalCare and Use Committee in accordance with appropriate institutional andregulatory bodies' guidelines.

Mice and Treatments

Eight- to 10-week-old specific-pathogen-free C57BL/6J mice werepurchased and allowed to acclimate for 2 weeks. Mice were fedstandardized laboratory rodent diet and sterile water ad libitum. Micewere randomly divided into the following 4 groups (n=5 mice/group): 1)NSAID (indomethacin); 2) indole; 3) NSAID+indole; and, 4) untreatedcontrols. Mice were then rehoused on the basis of treatment groupassignment, with 5 animals/group-cage. To induce NSAID enteropathy, micein group 1 were gavaged once daily with indomethacin (5 mg/kg; for 7days; Sigma Aldrich, St. Louis, Mo.) dissolved in dimethylysulfoxide(DMSO) (Sigma Aldrich, St. Louis, Mo.) and further diluted in phosphatebuffered saline (PBS). Mice in group 2 received indole by gavage (20mg/kg; once daily for 7 days; Sigma Aldrich, St. Louis, Mo.) dissolvedin sterile water warmed to 55° C. Mice in group 3 received indoleco-administered with indomethacin by gavage at the dosages describedabove. All mice were gavaged with equal volumes (200 μL) and equalconcentrations of DMSO (0.001%).

Sample Collection

Feces were collected daily by placing individual animals in sterileplastic cups that were RNase- and DNase-free until they passed feces.The mice were immediately returned to their home cages and the fecesimmediately flash frozen at −80° C. All animals were euthanized via CO₂asphyxiation on day 8 (i.e., after 7 days of treatment). The smallintestine was harvested, opened longitudinally, rinsed with ice-coldPBS, and the distal ⅓ of the intestinal mucosa was scraped for tissuegene expression analysis. The remaining small intestine was fixed in 4%paraformaldehyde, Swiss-rolled, paraffin-embedded, and stained withhematoxylin and eosin. The spleen and mesenteric lymph nodes (MLN) wereharvested, and immediately placed in ice cold RPMI-1640-c+10% fetal calfserum (FCS: Life Technologies, Carlsbad, Calif.), homogenized, andprepared as a single cell suspension for flow cytometric analysis aspreviously described (Wang N, Strugnell R, Wijburg O, Brodnicki T.Measuring bacterial load and immune responses in mice infected withListeria monocytogenes. Journal of Visualized Experiments: JoVE 2011).

Fecal Calprotectin ELISA

A murine calprotectin ELISA kit (HK214, Hycult Biotech, PlymouthMeeting, Pa.) was used according to the manufacturer's protocol withslight modifications. Briefly, 100 mg of feces was homogenized inextraction buffer (0.1 M Tris, 0.15 M NaCl, 1.0 M urea, 10 mM CaCl2, 0.1M citric acid monohydrate, 5 g/l bovine serum albumin (BSA) and 0.25 mMthimerosal [pH 8.0]). The homogenate was centrifuged at 10,000×g at 4°C. for 20 minutes and the supernatant used as directed in manufacturer'sprotocol.

Tissue RNA Extraction, Sequencing, and Processing

RNA was extracted from the mucosal scrapings using an RNeasy mini kit(QIAGEN, Redwood City, Calif.) following the manufacturer's instructionsand including on-column DNase treatment. RNA quantity was determinedusing a Nanodrop spectrophotometer (Fisher Thermoscientific) and thequality was assessed using the Nano6000 chip on a Bioanalyzer2100(Agilent Technologies). Only RNA with an integrity number (RIN)≥8was used. The samples were randomized before beginning the RNA-Seqlibrary preparation. Sequencing libraries were made using 250 ng of RNAand the TruSeq RNA Sample Preparation kit (Illumina) following themanufacturer's instructions. A volume of 2.5 μl of ERCC spike-in RNAcontrol mix (Life Technologies) was added to the starting RNA at adilution of 1:1000. The libraries were pooled and sequenced on anIllumina HiSeq 2500 at the Texas AgriLife Genomics and BioinformaticsServices Core Facility (College Station, Tex.). Sequencing data wereprovided in a de-multiplexed format and aligned using SplicedTranscripts Alignment to a Reference (STAR) software with defaultparameters and referenced against the genome of Mus musculus (Ensemblversion GRCm38) (Dobin A, Davis C A, Schlesinger F, Drenkow J, ZaleskiC, Jha S, Batut P, Chaisson M, Gingeras T R. STAR: ultrafast universalRNA-seq aligner. Bioinformatics (Oxford, England) 2013; 29:15-21).Differentially expressed genes were determined using EdgeR based on thematrix of gene counts (Robinson M D, McCarthy D J, Smyth G K. edgeR: aBioconductor package for differential expression analysis of digitalgene expression data. Bioinformatics (Oxford, England) 2010; 26:139-40).Gene lists were analyzed through use of QIAGEN's Ingenuity® PathwayAnalysis (IPA, QIAGEN, Redwood City, Calif.http://www.qiagen.com/ingenuity). Sequence data were uploaded into NCBIsmall reads archive (Accession number PRJNA290483).

Microbiota DNA Extraction, Sequencing, and Processing

Microbiota 16S rRNA gene sequencing methods were adapted from themethods developed for the NIH-Human Microbiome Project (A framework forhuman microbiome research. Nature 2012; 486:215-21; Structure, functionand diversity of the healthy human microbiome. Nature 2012; 486:207-14).Briefly, bacterial genomic DNA was extracted using MO BIO PowerSoil DNAIsolation Kit (MO BIO Laboratories) according to the manufacturer'sprotocol. The 16S rDNA V4 region was amplified by PCR and sequenced inthe MiSeq platform (Illumina) using the 2×250-bp paired-end protocolyielding paired-end reads that overlap almost completely (Caporaso J G,Lauber C L, Walters W A, Berg-Lyons D, Huntley J, Fierer N, Owens S M,Betley J, Fraser L, Bauer M, et al. Ultra-high-throughput microbialcommunity analysis on the Illumina HiSeq and MiSeq platforms. The ISMEJournal 2012; 6:1621-4). The primers used for amplification containadapters for MiSeq sequencing and dual-index barcodes so that the PCRproducts can be pooled and sequenced directly. The software suiteQuantitative Insights Into Microbial Ecology (QIIMEv1.9[http://qiime.sourceforge.net]) was used for data processing andanalysis (Caporaso J G, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello E K, Fierer N, Pena A G, Goodrich J K, Gordon J I, et al.QIIME allows analysis of high-throughput community sequencing data.Nature Methods 2010; 7:335-6). The raw sequence data werede-multiplexed, and low-quality reads were filtered using the database'sdefault parameters. Chimeric sequences were detected using Uchime andremoved prior to further analysis.⁶⁴ Sequences were then assigned tooperational taxonomic units (OTUs) using an open-reference OTU pickingprotocol [http://qiime.org/scripts/pick_open_reference_otus.html] withUCLUST software in QIIME based on 97% identity with the Greengenesdatabase (v13_5) (Edgar R C. Search and clustering orders of magnitudefaster than BLAST. Bioinformatics (Oxford, England) 2010; 26:2460-1;DeSantis T Z, Hugenholtz P, Larsen N, Rojas M, Brodie E L, Keller K,Huber T, Dalevi D, Hu P, Andersen G L. Greengenes, a chimera-checked 16SrRNA gene database and workbench compatible with ARB. Applied andEnvironmental Microbiology 2006; 72:5069-72; McDonald D, Price M N,Goodrich J, Nawrocki E P, DeSantis T Z, Probst A, Andersen G L, KnightR, Hugenholtz P. An improved Greengenes taxonomy with explicit ranks forecological and evolutionary analyses of bacteria and archaea. The ISMSJournal 2012; 6:610-8). To adjust for uneven sequencing depth among thesamples, each sample was rarefied to an even sequencing depth (10,512reads/sample) prior to further analysis.

Alpha rarefaction, beta diversity measures, richness, taxonomicsummaries, and tests for significance were calculated and plotted usingQIIME. The weighted and unweighted Unifrac distances were calculated forcomparison of beta diversity. Differences in microbial communities amongthe treatment groups were investigated by visual assessment ofclustering on principal component analysis (PCA) and by analysis ofsimilarity (ANOSIM) calculated on unweighted UniFrac distance metrics(Clark K. Non-parametric multivariate analyses of changes in communitystructure. Australian Journal of Ecology 1993; 18:117-43; Lozupone C,Knight R. UniFrac: a new phylogenetic method for comparing microbialcommunities. Applied and Environmental Microbiology 2005; 71:8228-35;Bray J R, Curtis J T. An ordination of upland forest communities ofsouthern Wisconsin. Ecological Monographs 1957:325-49). ANOSIM is anon-parametric test of difference between 2 or more groups based on adistance metric. This test gives an R value between −1 and 1 where largepositive R values indicate a large magnitude of dissimilarity betweenthe groups and small R values indicate small magnitudes ofdissimilarity; the P value provides statistical significance (Clark K.Non-parametric multivariate analyses of changes in community structure.Australian Journal of Ecology 1993; 18:117-43). When ANOSIM identifiedsignificant differences among groups, then pairwise ANOSIM was performedto determine which groups differed significantly and similaritypercentage (SIMPER) was used to examine which features contributed tothe differences among groups. ANOSIM, SIMPER, and PCA plots wereperformed with PAST v3.05 (Hammer O, Harper D, Ryan P D. PAST: PaleonStatictics Software Package for Education and Data Analysis.Palaeontologica Electronica 2001; 4:9).

The software Phylogenetic Investigation of Communities by Reconstructionof Unobserved States (PICRUSt) was used to predict the metagenome(Langille M G, Zaneveld J, Caporaso J G, McDonald D, Knights D, Reyes JA, Clemente J C, Burkepile D E, Vega Thurber R L, Knight R, et al.Predictive functional profiling of microbial communities using 16S rRNAmarker gene sequences. Nature Biotechnology 2013; 31:814-21). Sequencingdata were prepared as described above, but sequences were then clusteredinto OTUs using a closed-reference OTU picking protocol at the 97%sequencing identity level[http://qiime.org/scripts/pick_closed_reference_otus.html]. Theresulting OTU table was normalized by the expected copy number(s) of the16s rRNA gene in each OTU. PICRUSt was then used to predict themetagenome[https://picrust.github.io/picrust/tutorials/metagenome_prediction.html#metagenome-prediction-tutorial].Each sample was rarefied to an even sequencing depth to adjust foruneven sequencing depth prior to further analysis. Differences in themetagenomes among the groups were investigated by visual assessment ofclustering on PCA and by analysis of similarity (ANOSIM) calculated onBray Curtis dissimilarity metric.

Metabolite Extraction from Fecal Samples

Metabolites from the fecal contents were extracted using a solvent-basedmethod as previously described (Sridharan G V, Choi K, Klemashevich C,Wu C, Prabakaran D, Pan L B, Steinmeyer S, Mueller C, Yousofshahi M,Alaniz R C, et al. Prediction and quantification of bioactive microbiotametabolites in the mouse gut. Nature Communications 2014; 5:5492).Briefly, fecal pellets were homogenized using a homogenizer (OmniInternational) with equal volume of cold methanol and half volume ofchloroform. The samples were then centrifuged at 10,000 g at 4° C.(Thermo Fisher Scientific) for 10 min. Supernatant was passed through a70-μm sterile nylon cell strainer (Falcon) and 0.6 ml of ice cold waterwas added. The samples were vortex and centrifuged again at 10,000 g for5 minutes. The upper phase and lower phase were collected and 400 μl ofupper phase was dried to a pellet using a vacufuge (Eppendorf,Hauppauge, N.Y.), and then reconstituted in 50 μl of methanol/water(1:1, v/v). The samples were stored at −80° C. until analysis.Tryptophan metabolites in the samples were detected and quantified on atriple quadrupole linear ion trap mass spectrometer (3200 QTRAP, ABSCIEX, Foster City, Calif.) coupled to a binary pump HPLC (ProminenceLC-20, Shimazu, Concord, Ontario, Canada).

Flow Cytometry

Spleens and MLNs were processed individually to single-cell suspensionswith frosted glass slides in RPMI-c+10% FCS, and spleen cells underwentred blood cell lysis (Charles N, Hardwick D, Daugas E, Illei G G, RiveraJ. Basophils and the T helper 2 environment can promote the developmentof lupus nephritis. Nature Medicine 2010; 16:701-7). Cell suspensionswere plated in individual wells, washed with 0.5% BSA in PBS,surface-stained for CD11b-AlexaFluor488 (eBioscience cat. #53-0112-82)and Gr-1-biotin (BD cat. #553125), followed by streptavidin-PE(eBioscience cat. #12-4317), fixed with 0.4% paraformaldehyde, andsamples were acquired on a BD FACS Aria II in the College of MedicineCell Analysis Facility (COM-CAF) at the Texas A & M Health ScienceCenter.

Histology and small intestinal morphometric measurements

The stained sections of the small intestine were analyzed by aboard-certified veterinary pathologist (BRW) blinded to treatment group.The slides were scored as previously described for intestinalinflammation (Jia Q, Lupton J R, Smith R, Weeks B R, Callaway E,Davidson L A, Kim W, Fan Y Y, Yang P, Newman R A, et al. Reducedcolitis-associated colon cancer in Fat-1 (n-3 fatty acid desaturase)transgenic mice. Cancer Research 2008; 68:3985-91). Briefly, mucosalinjury was determined by the following parameters scored from 0 (noevidence) to 3 (marked): mucosal ulceration, mucosal erosion, andpresence of squamified epithelium. Inflammatory changes were scoredsimilarly based on the following parameters: lymphocytic infiltration,plasma cell infiltration, and neutrophilic infiltration. Finally, anoverall evidence of injury score was used to document total injurygraded from 0 (none) to 4 (marked). Morphological parameters wereobtained from digitally scanned slides using SPOT vr 5.0 software. Threesets of measurements from 3 separate sections were recorded for eachanimal by an observer blinded to treatment group. Measurements consistedof villus height, crypt depth, and submucosal mural thickness. The ratioof the villus height to crypt depth was calculated (FIGS. 6A and 6B)

Data Analysis

Results were expressed as mean±95% confidence interval unless indicatedotherwise. For all analyses, significance was set P≤0.05. Data wereanalyzed using S-PLUS statistical software (Version 8.2, TIBCO Inc.,Seattle, Wash.) unless otherwise noted. Histology scores, the proportionof neutrophils in the spleen and MLNs, ratios of villus height to cryptdepth, submucosal thicknesses, paired differences between Day 7 and Day0 in phyla and families, and fecal tryptophan metabolites were comparedamong treatment groups using a generalized linear model with post hoctesting for pairwise differences among groups using the method of Sidak(Šidák Z K. Rectangular confidence regions for the means of multivariatenormal distributions. J Am Stat Assoc 1967; 62:626-33). To meetstatistical assumptions underlying the generalized linear model, thehistology scores were converted to ranks and the neutrophil data werelog₁₀ transformed prior to analysis. Ratios of villus height to cryptdepth and submucosal thicknesses were compared among groups using ageneralized linear model with post hoc testing for pairwise differencesamong groups using the method of Sidak. Fecal calprotectinconcentrations were analyzed as a function of treatment group, time (Day0 [baseline] and Day 7), and their interaction using linearmixed-effects modeling with treatment group and time modeled as fixed,categorical effects and individual mouse modeled as a random effect toaccount for repeated measures on individual mice. Paired differencesbetween Day 0 and Day 7 in phyla and families were compared among groupsusing a generalized linear model and post hoc testing for pairwisedifferences among groups using the method of Sidak.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method of treating dysbiosis of commensal microbiota in a subject,comprising administering an effective amount of a tryptophan derivedmicrobiota metabolite (TDMM), or a precursor, prodrug, or acceptablesalt thereof.
 2. The method of claim 1, wherein the subject has receivedor is expected to receive administration of an agent suspected to causedysbiosis.
 3. The method of claim 2, wherein the agent causes areduction in a gram-positive component of the microbiota.
 4. The methodof claim 2, wherein the agent causes an increase in a gram-negativecomponent of the microbiota.
 5. The method of claim 2, wherein the agentis a non-steroid anti-inflammatory drug (NSAID).
 6. The method of claim1, wherein the TDMM is indole.
 7. The method of claim 2, wherein theTDMM, or a precursor, prodrug, or acceptable salt thereof, isco-administered with the agent.
 8. The method of claim 2, wherein theTDMM, or a precursor, prodrug, or acceptable salt thereof, isadministered in a pharmaceutical composition that also comprises theagent.
 9. The method of claim 1, wherein the TDMM is administered byintra-peritoneal (IP), intravenous (IV), topical, parenteral,intradermal, transdermal, oral (e.g., via liquid or pill), orrespiratory (e.g., intranasal mist) routes.
 10. The method of claim 1,wherein the subject is a mammal, such as a human or rodent.
 11. Themethod of claim 1, wherein treating dysbiosis prevents or amelioratesenteropathy.
 12. A method of treating enteropathy associated with NSAIDin a subject, comprising administering an effective amount of atryptophan derived microbiota metabolite (TDMM), or a precursor,prodrug, or acceptable salt thereof.
 13. The method of claim 12, whereinthe NSAID causes a reduction in a gram-positive component of themicrobiota.
 14. The method of claim 12, wherein the NSAID causes anincrease in a gram-negative component of the microbiota.
 15. The methodof claim 12, wherein the NSAID is selected from aspirin, salsalate,celecoxib, diclofenac, etodolac, ibuprofen, indomethacin, ketoprofen,ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sulindac,meloxicam, tolmetin, and the like.
 16. The method of claim 12, whereinthe TDMM is indole.
 17. The method of claim 16, wherein the effectiveamount of indole is at least about 5 mg/kg.
 18. The method of claim 12,wherein the TDMM, or a precursor, prodrug, or acceptable salt thereof,is co-administered with the NSAID.
 19. The method of claim 12, whereinthe TDMM, or a precursor, prodrug, or acceptable salt thereof, isadministered in a pharmaceutical composition that also comprises theNSAID.
 20. The method of claim 12, wherein the TDMM is administered byintra-peritoneal (IP), intravenous (IV), topical, parenteral,intradermal, transdermal, oral (e.g., via liquid or pill), orrespiratory (e.g., intranasal mist) routes.
 21. The method of claim 12,wherein the subject is a mammal, such as a human or rodent.
 22. A methodof treating a condition characterized by inflammation in the GI tract,comprising administering an effective amount of a tryptophan derivedmicrobiota metabolite (TDMM), or a precursor, prodrug, or acceptablesalt thereof, to a subject in need thereof.
 23. The method of claim 22,wherein the inflammation is associated with administration of an NSAIDto the subject.
 24. A pharmaceutical composition comprising: at leastone TDMM, or a precursor, prodrug, or acceptable salt thereof; at leastone NSAID composition, or a precursor, prodrug, or acceptable saltthereof; and a pharmaceutically acceptable carrier.
 25. Thepharmaceutical composition of claim 24, wherein the TDMM is indole.